The long term goal of this project is to determine the molecular mechanisms that underlie neurotransmission. The goal of the next project period is to understand the role of clathrin-mediated vesicle recycling in neurotransmission as well as to better understand the molecular mechanisms of clathrin-coat assembly and disassembly. Aim 1 tests the hypothesis that synaptic vesicle coating and uncoating are required for normal neurotransmission by examining the effects of reagents that disrupt clathrin assembly and vesicle uncoating on neurotransmission in the squid giant synapse preparation. Aim 2 will determine whether synaptic vesicle proteins participate in the formation of clathrin coats. A combination of biochemical and physiological techniques will be used to critically evaluate the proposed roles of the synaptic vesicle proteins synaptotagmin, synaptobrevin and SV2 as well as a novel protein identified in a yeast two hybrid screen for proteins that bind the clathrin adaptor protein AP180. Aim 3 will assess the physiological importance of two regions of the adaptor protein AP180, the clathrin assembly domain and the inositol lipid-binding domain, by testing these activities in C. elegans AP180 mutant proteins and by assessing the ability of novel mutant proteins to rescue and AP180 mutant phenotype. Aim 4 will critically evaluate the hypothesis that interactions between resident coat proteins regulate the efficiency of synaptic vesicle endocytosis utilizing a combination of biochemical and genetic approaches. Studies will focus on interactions between AP180, AP2, eps15 and auxilin. Aim 5 will biochemically characterize novel C. elegans AP180 mutants that have defects in synaptic transmission. Successful completion of these studies will allow us to achieve a deeper level of understanding of the processes of endocytosis and neurotransmission.